The other eukaryal-like
replication proteins than can be readily
identified in the P. abyssi proteome are the MCM
helicase (PAB2373),
DNA pol I (PAB1128),
RNAse H II (PAB0352),
the two subunits of DNA primase
(PAB2235,
PAB2236),
the three subunits of RP-A (RP-A39:
PAB2163,
RP-A14: PAB2164
and RP-A21: PAB2165),
the PCNA clamp (PAB1465),
the flap-endonuclease FEN 1
(PAB1877)
and DNA ligase (PAB2002).

Transcription

In general, promoter
regions are A/T rich to facilitate local
unwinding of the DNA helix upon transcription
initiation. In addition, the A/T enrichment of
archaeal intergenic regions can be attributed to
the presence of TATA box and transcription
factor B recognition elements (BRE): the
consensus sequence of 19 mapped promoters of
Pyrococcus furiosus has a similar A/T bias (at
position -36/-23 relative to the transcription
start site:
[G/A],A,A,A,N,N,T,T,[A/T],[A/T],[A/T],[A/T],A)
(Verhees 2002). The predicted non coding regions
in the Pyrococcus genome indeed display a strong
over-representation of 6 trinucleotides (AAA,
AAT, ATT, TAA, TTA and TTT) whereas coding
regions do not exhibit any clear-cut bias in
overall trinucleotides composition (see figure).

Frequencies of all 64
trinucleotides have been computed for complete
genome, coding and intergenic regions. The ratio
of observed (%Obs) to full genome frequencies
(%total) is displayed in the
histogram.

Motility

The membrane potential
is used to drive the flagellar motor. Like other
Pyrococci, P. abyssi has typical archaeal
flagella. The flagellar
operon
contains 3 copies of the flagellin gene flaB
(PAB1378-PAB1380),
followed by flagellar accessory genes
flaCDEFGHIJ (PAB1381-PAB1387).

Although archaeal
flagella differ from bacterial ones in both
structure and composition (Thomas et al., 2001),
several flagellar assembly proteins such as the
ATPase FlaI (PAB1386)
and the signal peptidase FlaK
(PAB1309)
show similarity to proteins involved in the
biogenesis of type IV pili in bacteria (Bardy
and Jarrell, 2002). A set of methyl-accepting
chemotaxis proteins has been identified in the
genome (PAB1026-PAB1027,
PAB1330-PAB1336).

The fact that this
operon is also present in P. horikoshii but not
in P. furiosus, has been suggested to correlate
with the wider range of biosynthetic pathways
encoded by the latter genome, which might
obviate the need for the chemotactic response
(Maeder et al., 1999).

Amino acid
biosynthesis

The entire set of
enzymes involved in the 10-step tryptophan
biosynthesis pathway is encoded in the P. abyssi
GE5 genome. The first steps of this pathway,
leading from phosphoenolpyruvate and
erythrose-4-phosphate to chorismate, are encoded
in a single aroGBDEKP---AC putative
operon
(PAB0297-PAB0307),
which additionally includes genes for an
ABC-type transport system of unknown substrate
specificity. In P. abyssi the genes encoding
chorismate mutase and prephenate dehydrogenase,
required for Phe and Tyr synthesis are missing
(Table ).

The enzymes that
constitute the classical bacterial lysine
biosynthesis pathway are not encoded by the P.
abyssi genome. However, the gene
cluster
encoding an alternative route, the
alpha-aminoadipic acid (AAA) pathway that was
recently characterised in the thermophilic
bacterium Thermus thermophilus (Nishida et al.,
1999), appears to be conserved in P. abyssi, as
well as in P. horikoshii and P. furiosus. The
lys operon encodes enzymes that resemble their
counterparts in leucine and arginine synthesis:
leuACDB, argCBDE, a small ORF with similarity to
a zinc ribbon, and a RimK-like gene which has
previously been proposed to code for an
ATP-dependent carboxylate-amine/thiol ligase
(Koonin et al., 1997). In P. furiosus it has
recently been proposed that these genes are also
involved in the synthesis of ornithine, which is
converted in 3 steps to arginine by ArgFGH
(Brinkman et al., 2002). Unlike P. furiosus,
however, P. abyssi appears not to possess
argFGH, in agreement with its arginine
auxotrophy (Table).

All the enzymes
necessary for the synthesis of threonine are
present. Aspartate kinase is represented by two
genes (PAB1674
and PAB1675)
indicating a possible different regulation of
threonine and methionine biosynthesis, since the
product of aspartokinase, aspartylphosphate is a
common precursor of these two amino acids. Genes
for three other enzymes of threonine
biosynthesis, aspartate semialdehyde
dehydrogenase (PAB1678),
homoserine kinase (PAB1676)
and threonine synthase (PAB1677),
are clustered with the aspartate kinase genes,
forming a probable
operon,
whereas the homoserine dehydrogenase gene
(PAB0610)
is located next to several genes of
methionine
biosynthesis
(PAB0605-PAB0608).
The enzymes of the branch leading from threonine
to isoleucine are found, with the exception of
the small regulatory subunit of
aceto-hydroxyacid synthase. The enzymes common
to valine and isoleucine biosynthesis are
present in P. abyssi as well as the threonine
dehydratase, specific to isoleucine synthesis.
The genes encoding the common isoleucine/valine
biosynthesis enzymes are clustered
with
the genes of leucine biosynthesis
(PAB0888
to PAB0895,
including PAB2424)
(Table ).

All the enzymes
required for serine, glycine and cysteine
biosynthesis are present in P. abyssi, with the
exception of PAPS phosphotransferase and
sulphite reductase. Of the two subunits of
bacterial NADPH-dependent glutamate synthase,
the large subunit (equivalent to the E. coli
GltB and B. subtilis GltA) is missing in P.
abyssi, while the small subunit (GltD in E.
coli) is encoded in two copies
(PAB1738
and PAB1214).
In Pyrococcus kodakaraensis (Jongsareejit et
al., 1997), a homotetramer of this subunit was
found to be capable of both glutamine-dependent
and ammonia-dependent synthesis of glutamate
without the presence of an equivalent of the E.
coli large subunit. The glutamine synthetase
gene (PAB1292)
is present in P. abyssi. Although no protein
catalysing a covalent modification has been
detected, it should be noted that the tyrosine
residue on which the adenylylation occurs in E.
coli as well as six surrounding residues are
conserved in the P. abyssi sequence. Several
aminotransferases have been detected, catalysing
the final steps of valine, leucine and
isoleucine synthases, as well as aspartate and
glutamate synthesis from oxaloacetate and
2-oxoglutarate. In addition P. abyssi possesses
an aromatic amino acid aminotransferase, several
omega amino acid aminotransferases and a
serine-glyoxylate aminotransferase.

Although P. abyssi has
been found to be methionine auxothroph, several
orthologs of bacterial enzymes involved in
methionine biosynthesis are present, with the
notable exception of homoserine acyltransferase
and methyltetrahydrofolate reductase. The
B12-dependent variant of methionine synthetase
is not encoded in P. abyssi, but B12-independent
enzyme, methylating homocysteine into methionine
is found in two copies (PAB0608
and PAB2361).
Also found are the archaeal type
S-adenosylmethionine synthetase
(PAB2094)
and a S-adenosylhomocysteinase
(PAB1372),
which is probably involved in a methionine
salvage pathway. Despite the reported proline
prototrophy, none of the classical proteins
responsible for proline biosynthesis has been
detected in P. abyssi GE5 genome, raising the
possibility of an unique proline synthesis route
for Pyrococci (Fig. 1, text).

Amino
acid biosynthesis

Gene
name

(predicted)
PAB

Phe, Tyr
biosynthesis

pheA/aroH

No

tyrA

No

aspC

No

Trp
biosynthesis

aro-operon

297-307

trp-operon

2043-2049

His
biosynthesis

his
operon

No

Ser
biosynthesis

serAB

514,1207

Gly
biosynthesis

glyA

2018

Thr
biosynthesis*

thr-operon

1674-1678

Cys
biosynthesis

cysKM

250,605

Leu
biosynthesis*

leuABCD

890-894,2424

Ile, Val
biosynthesis*

ilvBCD

888,889,895

Met
biosynthesis*

605-608,610,1372,2094,2361

Pro
biosynthesis**

novel type
?

?

Lys
biosynthesis (AAA-type)

lysYZJK

286-294

Arg
biosynthesis

argGH

No

Ala
biosynthesis

alaAT

1810

Asp
biosynthesis

aspAT

Several
ATs

Glu
biosynthesis

gltD

1214,1738

Gln
biosynthesis

glnA

1292

Asn
biosynthesis

asnB

750,1605

Nucleotides
& cofactors

Present

Purine
biosynthesis*

Yes

Pyrimidine
biosynthesis

Yes

NAD
biosynthesis

Yes

Heme
biosynthesis

No

Cobalamin
biosynthesis

No

Folate
biosynthesis

No

Pyridoxal
biosynthesis

No

Biotin
biosynthesis

No

Coenzyme A
biosynthesis*

Yes

Heme
biosynthesis

No

Anabolic capacity of
P. abyssi as deduced from genome
analysis.
Predicted genes/operons involved in amino acid
biosynthesis are indicated by PAB identifier;
when no gene has been identified it is indicated
(No). In some cases there is a discrepancy with
experimentally-determined autotrophy (*) or
prototrophy (**).

Vitamin
biosynthesis

P. abyssi does not
encode enzymes of the biotin biosynthesis and
has to import it through a still uncharacterised
transport system. It encodes, however, two
copies of biotin-(acetyl-CoA carboxylase)
ligase, which links biotin to the
biotin-carboxyl carrier protein. One of these
two copies is fused in a bi-functional protein
BirA (PAB0104)
to the biotin-dependent transcriptional
regulator, as is the case in many bacteria. In
contrast of P. furiosus, P. abyssi and
P.horikoshii do not encode enzymes of riboflavin
biosynthesis and we were not able to identify
transporters involved in flavin uptake.

P. abyssi encodes
transketolase and acetolactate synthase, the
thiamine diphosphate-dependent enzymes, and
apparently can both synthesise thiamine and
acquire it from the outside. Indeed, P. abyssi
encodes a putative ABC-type transport system,
consisting of adjacent genes for a
thiamine-binding periplasmic protein
(PAB1835),
an ATPase (PAB0545),
and a permease (PAB0543).
In addition, P. abyssi encodes homologs of most
(but not all) bacterial enzymes of thiamine
biosynthesis (ThiC, PAB1930;
ThiD, PAB1646;
ThiE, PAB1645;
ThiL, PAB2358).
In contrast, the enzymes involved in the
synthesis of the thiazole moiety in bacteria are
only partly represented in P. abyssi. This could
be due to the fact that bacteria produce
thiazole ring from 1-deoxy-D-xylulose
5-phosphate, which appears to be a
bacteria-specific sugar. The sugar that serves
as thiazole precursor in archaea remains
unknown, but the proteins responsible for the
introduction of sulphur into the molecule (ThiS,
PABs5591;
ThiF, PAB2302;
and ThiI, PAB0226
and PAB0561)
seem to be the same. It should be noted that as
compared to bacterial ThiD, PAB1646
and all other archaeal homologs of ThiD contain
an additional 180-aa C-terminal domain of
unknown function that is most likely involved in
thiamine biosynthesis.

P. abyssi encodes a
complete set of enzymes of pyridine nucleotide
biosynthesis (NadA, PAB2345;
NadB, PAB2343;
NadC, PAB2347).
The apparent absence of 1-deoxy-D-xylulose
5-phosphate in archaea (see above) suggests that
pyridoxine ring is formed from some other sugar
by products of the PDX1 and PDX2 (formerly
SNZ/SNO) genes, described in Cercospora
nicotianae and in yeast (Ehrenshaft and Daub,
2001). These two genes (PAB0537
and PAB0538)
are the only pyridoxine biosynthesis genes found
in P. abyssi.

P. abyssi genome does
not contain heme biosynthesis related genes.
However, genes encoding enzymes for the last
steps of adenosylcobalamin biosynthesis have
been detected: cobalamin biosynthesis protein
CbiB (PAB0025),
cobalamin-5-phosphate synthase(CobS,
PAB2320),
ATP:corrinoid adenosyltransferase (BtuR,
PAB2289),
and nicotinic acid
mononucleotide:5,6-dimethylbenzimidazole
phosphoribosyltransferase (CobT,
PAB2326).
It appears that P. abyssi would have to import a
corrinoid precursor of the cobalamine.

Isoprenoid
biosynthesis and utilization

Acetyl CoA
C-acetyltransferase (PAB0907),
3-hydroxy-3-methylglutaryl CoA synthase
(PAB0906)
and reductase (PAB2106)
as well as mevalonate kinase
(PAB0372)
are found in P.abyssi, in accordance with all
Archaea investigated to date. The orthologs of
the proteins, accounting for the route from
mevalonate to pyrophosphomevalonate are missing,
as are the enzymes of the alternative
deoxy-D-xylulose phosphate synthase pathway
(Smit and Mushegian, 2000). Whatever is the
source of pyrophosphomevalonate, an
isopentenyldiphosphate isomerase is synthesised
by P. abyssi (PAB1662)
as well as a multifunctional
isoprenyldiphosphate synthase
(PAB2389),
a homolog of an enzyme from Archaeoglobus
fulgidus that catalyses the synthesis of
geranylgeranyldiphosphate (Wang et al., 1999). A
polyprenyldiphosphate synthase
(PAB0394)
has been detected. as well as a geranylgeranyl
hydrogenase (PAB01O9),
known to be a precursor of phytol, component of
chlorophylls.

Ehrenshaft, M., and
Daub, M.E. (2001)
Isolation of PDX2, a second novel gene in the
pyridoxine biosynthesis pathway of eukaryotes,
archaebacteria, and a subset of eubacteria.
J Bacteriol 183: 3383-3390.